Cyclic GMP is increased markedly in fat cells within seconds after the addition of catecholamines. Since this increase in cyclic GMP cannot be attributed to direct activation of guanylate cyclase we have examined the possibility of the activation of the enzyme by calcium and other divalent metal ions. It has been shown earlier that efflux of calcium is markedly increased just prior to the increase in cyclic GMP and the mobilization of calcium may be responsible for the increase in cyclic GMP. A large portion of guanylate cyclase in fat cells appears to be associated with cell membrane fraction. The enzyme activity is linear with time (30 min) and protein concentration. The substrate for enzyme reaction appears to be MnGTP. For optimamal enzyme activity Mn ions are required, 50% maximal activation is obtained at 1 mM free Mn ions. Mg ions and Ca ions are able to replace Mn ions but are much less effective. When the enzyme is assayed with 100 micron M GTP, the enzyme activity is markedly potentiated by low concentrations of Ca ions (1-100 micron M) in combination with low concentration of Mn ions. A similar potentiation by Ca ions is also seen. Mn ions are replaced by Mg ions.